The invention relates to apparatus for the separation of macromolecules by electrophoresis, particularly by isoelectric focusing.
Two-dimensional (2D) electrophoresis is a useful and well-known separation technique for purifying macromolecules and separating complex macromolecule mixtures, often providing a much higher resolving power than typical electrophoretic separation in one dimension or direction.
In two-dimensional electrophoresis, separation is performed under two different conditions or according to two different separation parameters, one in a first direction and the other in a second direction which is usually perpendicular to the first. The first dimension of the separation is typically performed in an elongate rod-shaped or strip-shaped gel with migration and separation of macromolecules occurring along the length of the gel. Once the macromolecules have been grouped into individual zones along the length of the gel, the gel is placed along one edge of a slab gel and the electric current is imposed across the both gels in a direction perpendicular or otherwise transverse to the first (elongate) gel. This causes the migration of the macromolecules from each zone of the elongate gel into the slab gel, and the separation of macromolecules within each zone.
A variety of combinations can be used for the first and second dimension separations. Separation based on charge or pI can be followed by separation based on molecular weight, for example. Likewise, separation in a gel of one concentration can be followed by separation in a gel of the same material but of another concentration. Two-dimensional separations have also been used to create a stepwise change in pH to separate first in a homogeneous gel and then in a pore-gradient gel, to separate in media containing first one protein solubiliser and then another, or in media containing a protein solubiliser first at one concentration and then at another concentration, to separate first in a discontinuous buffer system and then in a continuous buffer system, and to separate first by isoelectric focusing and then by homogeneous or pore gradient electrophoresis. Combinations such as these can be used to separate many kinds of macromolecules, including serum or cell proteins, bacterial proteins, non-histone chromatin proteins, ribosomal proteins, mixtures of ribonucleo-proteins and ribosomal proteins, glycoproteins, and nucleic acids.
There are many difficulties and disadvantages with existing apparatus suitable for running immobilised pH gradients (IPGs). There is no possibility of rehydrating the IPGs in the apparatus. All commercially available systems have electrodes which are removable and therefore can be placed incorrectly in use. Many of the electrodes are resilient which can result in poor electrical contact. Due the size and configuration of the apparatus available, they require a relatively large quantity of paraffin oil (xcx9c250 mL). This not only results in the expense of discarding the oil but also requires intense washing between runs and after each use. Due the inherent design of current systems they have a relatively large footprint and thus require a large amount of bench space in use.
Another disadvantage of two-dimensional electrophoresis systems is that they are labor-intensive and time-consuming. Elongate gels are awkward to handle and their use is prone to error and loss of samples and time. The present inventors have now developed a new electrophoresis apparatus that is useful for running IPGs.
In a first aspect, the present invention consists in an electrophoresis apparatus for the simultaneous electrophoretic separation of a plurality of component mixtures, the apparatus comprising:
a tray which is detachable from and removable from the apparatus, the tray defining a plurality of troughs, each trough being shaped to retain an elongate strip of isoelectric focusing medium and sufficient liquid to immerse the elongate strip;
a frame to retain the tray in a predetermined position;
a pair of electrodes adapted to be positioned at or near each end of the tray, wherein the electrodes are arranged such that each electrode is aligned with each of the plurality of troughs at the respective ends of the tray; and
electrically conductive connection means for connecting to a means for imposing an electric potential between the electrodes.
In one preferred form, the tray is provided with the electrodes positioned in the troughs and having electrical terminals positioned at one end of the leads. When the tray is placed in the frame, the terminals connect to plugs positioned therein thus allowing the imposing of an electric potential between the leads.
A principal advantage of the present invention is that it allows the tray to be disposable although trays could be provided for multiple use, if desired. In the case of disposable trays containing electrodes any suitable conductive material compatible with electrophoresis could be used as the electrode although clearly cheaper electrodes such s graphite would be preferable to platinum if the tray is to be thrown away after use.
Preferably, the tray is fabricated of a heat-transmissive material to permit temperature control of the troughs through the tray. In one form, the tray comprises 3 to 24, preferably 6 to 18, substantially parallel troughs. The troughs are preferably of equal length and each from 6 cm to 20 cm in length. It will be appreciated, however, that the tray may have any number of troughs, and the troughs be any suitable length and depth.
The apparatus according to the first aspect of the present invention also allows the possibility to stack a plurality of trays and carry out multiple separations simultaneously. The trays are removable for loading etc, and then would simply plug in to a suitable frame adapted to receive a number of trays stacked horizontally. Thus one power supply could be used for a number of trays.
Alternatively, several apparatus containing the frame with the tray attached may be stacked to carry out simultaneous separations.
In use, each elongate strip of isoelectric focusing medium placed in a trough will be subjected to the electric potential.
The electrically conductive connecting means may be electrical leads attachable to the electrodes for connecting the electrodes to a suitable power source.
In a related aspect, the present invention consists in an electrophoresis apparatus for the simultaneous electrophoretic separation of a plurality of component mixtures, the apparatus comprising:
a detachable tray containing a plurality of troughs, each trough shaped to retain an elongate strip of isoelectric focusing medium and sufficient liquid to immerse the elongate strip:
a frame to retain the tray in a substantially horizontal position;
a pair of support members rotatably mounted to the frame such that when the tray is retained therein each support member spans the plurality of troughs, each the support member supporting a plurality of electrodes arranged such that one electrode from each support member is aligned with each of the plurality of troughs;
means for rotating each support member between a first position in which all electrodes thereon extend into the troughs and a second position in which all electrodes thereon are clear of the troughs; and
electrically conductive connection means for connecting to a means for imposing an electric potential between the electrodes on one support member and the electrodes on the other support member.
Preferably, each of the support members has an axis of rotation that is transverse to the parallel troughs. The apparatus may further include a means for biasing each the support member into the first position. The support members, in addition to being rotatably mounted to the frame, may also be movably mounted thereto at a variable distance from each other.
Preferably, the tray is fabricated of a heat-transmissive material to permit temperature control of the troughs through the tray. In one form, the tray comprises 3 to 24, preferably 6 to 18, substantially parallel troughs. The troughs are preferably of equal length and each from 6 cm to 20 cm in length.
The electrically conductive connecting means may be electrical leads attachable to the electrodes for connecting the electrodes to a suitable power source.
In a third aspect, the present invention consists in a method of separating a macromolecule comprising subjecting the macromolecule to electrophoresis in an apparatus according to the first or second aspects of the present invention.
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following examples and accompanying drawings.